Method for supplying oxygen to a water purification process

ABSTRACT

The present invention relates to a method for supplying oxygen to a water purification process, said method comprising providing an oxygen carrier of at least one copolymer of dimethylsiloxane, ethylene oxide and propylene oxide, adding said oxygen carrier to the water purifying process, and contacting said oxygen carrier with an oxygen containing gas. The invention further relates to the use of at least one copolymer of dimethylsiloxane, ethylene oxide and propylene oxide, as an oxygen carrier in a water purification process.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a method for supplying oxygen to awater purification process and to the use of at least one copolymer ofdimethylsiloxane, ethylene oxide and propylene oxide, as an oxygencarrier in a water purification process.

BACKGROUND ART

The processing and disposal of wastewater treatment sludge areincreasingly important topics of environmental, economical andtechnological concern. Recently, the waste volumes produced haveincreased dramatically as a result of increases in the organic loadingof waste-water and environmental regulations that require a higherdegree of wastewater treatment. After sewage treatment at wastewaterplants there is still over 1 million ton sludge produced each year inSweden. By tradition, this sludge has been spread out on fields asfertilizer, or it has been deposited or combusted. However, manyproblems have arisen. In 1999 Lantbrukarnas Riksförbund, the farmer'snational union in Sweden, warned their members from using sludge asfertilizer as they suspected the sludge to contain hazardous substances.Disposal by land filling is also becoming increasingly expensive. Thegrowing and closing of landfills, public concerns over ground-watercontamination and safety problems associated with methane production asa result of biological activity in landfills further expand the problem.Public concern over possible hazardous products through combustionprocesses such as dioxins and possible heavy metal contamination fromthe resulting ash is also problematic. Therefore a new waste tax wasintroduced in January 2002 to encourage researchers to find a bettersolution. By year 2005, the situation becomes even more critical as bythen it will be completely illegal to deposit organic material assludge. Therefore, the problem with the great amount of sludge mass isurgent today.

Certain wastewater treatment methods comprise biological techniques,such as aerobic treatment, anaerobic treatment, and other anoxicprocesses (denitrification, sulphate reduction). The biologicaltechniques clearly have the greatest potential for treating wastewater.Biological processes can be used to remove and/or recover biodegradableorganic compounds, nitrogen, phosphorus and sulphuric compounds,pathogenic organisms and various heavy metals.

Biological processes are used extensively in the treatment of domesticand industrial wastewater. The quality of the effluent water depends oneffective removal of the pollutants by metabolic activity of the aerobicmicroorganisms. The activity depends on the growth rate which isregulated by the dissolved oxygen and medium composition. Aerobicconditions are favouring oxidation of substances responsible for theunpleasant odour of fermented sludge.

Oxygen solubility has always been an important issue in many aerobicfermentation processes, because oxygen unlike other nutrients issparingly soluble in aqueous media. The mass transfer rate of oxygenfrom oxygen rich phase to media is often a rate limiting factor in theprocesses. Therefore oxygen has to be continuously supplied to media tomeet the oxygen demand required for actively respiring cells to do themetabolism which will not be effected by the lack of oxygen. Shortage ofdissolved oxygen is hampering the biological digestion of sludge andoxidation reaction e.g. nitrification and detoxication of wastewater.Bioremediation of hazardous toxicants such as dioxins and pesticides isoxygen dependant.

Today the lack of oxygen in the biological purification steps ispartially solved by supplying oxygen by pumping to these biologicalsteps. This requires a high energy supply which is expensive. However,since there still remains a considerable amount of sludge, which must bedeposited and discarded, new means for supplying oxygen to thebiological steps are required, so that a more effective digestion isaccomplished.

In WO86/03773 there is described a process for increasing the solubilityof gases in an aqueous medium and an emulsion for carrying out saidprocess. Said emulsion comprises a copolymer of a silicone and ahydrophilic compound.

Due to the restricted laws regarding disposal of organic material assludge in the coming years there are urgent needs to develop new meansto digest sludge more efficiently. The present invention provides asolution to the above mentioned problem.

SUMMARY OF THE INVENTION

The present invention relates, in one aspect, to a method for supplyingoxygen to a water purification process, said method comprising:

a) providing an oxygen carrier of at least one copolymer ofdimethylsiloxane, ethylene oxide and propylene oxide;

b) adding said oxygen carrier to the water purifying process; and

c) contacting said oxygen carrier with an oxygen containing gas.

The invention relates, in another aspect, to the use of at least onecopolymer of dimethylsiloxane, ethylene oxide and propylene oxide, as anoxygen carrier in a water purification process.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

In an embodiment of the invention said copolymer is added as anemulsion, or as a copolymer immobilized on and/or in a support. Anemulsion may be used for example for increasing the oxygen content ofcontaminated waste water, thereby increasing the digestion of heaviermaterial such as sludge. The copolymer may be emulsified in any solventknown to a person skilled in the art, an example being water or any oil.A support immobilized with said copolymer may be used for increasing theoxygen content for bioremediation of the waste.

The oxygen carrier of at least one copolymer of dimethylsiloxane,ethylene oxide and propylene oxide added to the water purifying processmay be combined with other known means for increasing the oxygen contentin water. A certain water purifying process is not limited to a certaincopolymer, but a combination of different copolymers ofdimethylsiloxane, ethylene oxide and propylene oxide may be addedtogether or separately to the water purification process.

In a further embodiment said copolymer immobilized on a support furtherincludes immobilized microorganisms thereon. Said support may beselected from the group consisting of organic supports, such asalginate, collagen, glycans, and so on, and non-organic supports, suchas ceramics, polystyrene in the form of hollow fiber membranes, and thesurfaces of beads. Any support known within the art may be used inconnection with the invention and will be apparent to a person skilledin the art. By co-immobilizing the copolymer with microorganisms andoxidative enzymes participating in aerobic processes, the contact of theoxygen carrying copolymer and the microorganisms and oxidative enzymesis facilitated, thereby getting easy access to the required oxygen. Theoxidation process is accelerated.

In a yet further embodiment said oxygen containing gas is added to theprocess either continuously or batch-wise. By continuously adding oxygento the process the copolymer can continue to function as an oxygencarrier over time. As the oxygen is consumed by the aerobic processesfurther oxygen can be continued to be supplied to the copolymer. Thesupplied oxygen gets into contact with the copolymer, being depleted ofoxygen, and the copolymer takes up oxygen and continues to act as anoxygen carrier.

In the context of the present invention the wording “oxygen containinggas” refers to any kind of gas containing oxygen, examples being air orpure oxygen gas.

In an embodiment of the invention said copolymer is added to the aerobicsteps of the water purifying process. In the present context the wording“aerobic step(s)” is meant to comprise any step(s) of a waterpurification process in a water purification plant requiring oxygen forthe digestion of sludge or any contaminants in the waste water.

In another embodiment of the invention said at least one copolymercomprises 10-40% by weight of dimethylsiloxane, 20-60% by weight ofethylene oxide, and 10-60% by weight of propylene oxide.

In a yet further embodiment said copolymer comprises 15-35% by weight ofdimethylsiloxane, 25-45% by weight of ethylene oxide and 20-50% byweight of propylene oxide. Non-limiting examples of copolymers whichpresent satisfactory results in connection with the present inventionare a copolymer comprising 18% by weight dimetylsiloxane, 35% by weightethylene oxide and 46% by weight propylene oxide and a copolymercomprising 33% by weight dimetylsiloxane, 44% by weight ethylene oxideand 23% by weight propylene oxide.

The copolymers are used for supplying the desirable oxygen amount to themicroorganisms requiring oxygen for their metabolism, i.e. digestion ofsludge. Further, the copolymers are used for transporting other gases,e.g. carbon dioxide, produced in the digestion of the sludge. Thecopolymers may be added to the aerobic steps of a water purificationprocess in any form, examples being as an emulsion or immobilized on asupport. Any form which suits the copolymer may naturally be used in thescope of the present invention. The amount required is generally low andthe copolymer is biodegradable. Thus, a further advantage of theinvention is that the copolymer degrades after some time and it is notnecessary to remove the remainders from the process. Thus, it is onlyrequired to add further copolymer to the water purifying process iffurther purification is needed.

Both agitation and air compression, being used today for supplyingoxygen, consume a considerable amount of energy. Therefore, copolymersof dimethylsiloxane, ethylene oxide and propylene oxide can serve as acheap solution to enhance oxygen levels in aerobic steps in waterpurification. It is important to select a proper copolymer ofdimethylsiloxane, ethylene oxide and propylene oxide, since it has beenshown in the present invention that certain copolymers supply oxygenmore efficiently than others.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Oxidation rate of different concentrations of glucose byimmobilised glucose oxidase, monitored by thermometric sensor. The Yaxis represents the glucose oxidase activity (delta H/min) and the Xaxis represents the concentration of glucose.

FIG. 2. The effect of the polydimethylsiloxane (PDMS) copolymers, withincreasing concentrations of PDMS, on the enzymatic glucose oxidation.The Y axis represents the glucose oxidase activity (Delta H/min) and theX axis represents the identity of the composition. The tested samplesare in table 1. In each analyzed sample glucose (2.5 mM) was present.Symbols: 1) no polymer; 2) 15% DC Q2-5247; 3) 0.5% DC1248+15% DCQ2-5247; 4) 5% DC1248+15% DC Q2-5247; 5) 15% DC 1598 ; 6) 0.5%DC1248+15% DC 1598 ; 7) 5% DC1248+15% DC 1598; 8) 15% DC1248+0.5% PPGP2000.

FIG. 3. The role the poly ethylene/propylene block in the PDMS copolymerhas on the enzymatic glucose oxidation. The Y axis represents theglucose oxidase activity (Delta H/min) and the X axis represents theidentity of the composition. In each analyzed sample glucose (2.5 mM)was present. Symbols: 1) no polymer ; 2) 15% DC Q2-5247; 3) 15% DCQ2-5247+5% pluronic F 68; 4) 15% DC 1598; 5) 15% DC 1598+5% pluronic F68.

FIG. 4. Enzymatic glucose oxidation in the presence ofpolydimethylsiloxane (PDMS) copolymers and Perfluorodecalin. The Y axisrepresents the glucose oxidase activity (Delta H/min) and the X axisrepresents the identity of the composition. Symbols: 1) no polymer; 2)15% perfluorodecalin +5% pluronic F 68; 3) 15% DC 1598+5% pluronic F 68;4) 15% DC Q2-5247+5% pluronic F 68; 5) 15% DC 1248+0.5% PPG P2000

FIG. 5. Growth curve of Bacillus thuriginensis with and without DCQ2-5247 added into LB growth medium. The Y axis represents CFU/ml ofBacillus thuriginensis (coloni forming units/ml) and the X axisrepresents the time lapsed (hours). The respective curves represent acontrol (no copolymer added), 0.05% of PDMS copolymer (Q2-5247) and 0.1%of PDMS copolymer (Q2-5247).

EXAMPLES MATERIAL AND METHODS

Preparation of Thermistor Based Enzyme Unit

Glucose oxidase (GO) with horseradish peroxidase (HRP) were covalentlyco-immobilized on amino-controlled-pore glass beads (CPG) by usingglutaraldehyde chemistry. CPG was activated with 2.5% glutaraldehydesolution dissolved in phosphate buffer (0.1N, pH 7.0). To 350 mg ofactivated CPG beads were added 10 U of dialyzed glucose oxidase andperoxidase in phosphate buffer. The coupling of the enzymes was carriedout overnight at 4° C. under gentle shaking. Afterwards, in order toterminate all the unreacted groups on the matrix 50 mg ethanol amine (pH8.5-9.0) was added and allowed to react further for 2 h. Finally, theimmobilised enzymes were washed with the (10 vol ) phosphate buffer andthe beads were transferred into a column (0.7 ml, 2.5 cm×0.7 cm) andassembled into the thermometric system belonging to the unit calledenzyme thermistor (ET).

Assay

The reaction velocity is determined by converting the produced heatduring the enzymatic oxidation of glucose into electrical signals readas peaks. The size of the integrated peak area is proportional to theconcentration of the oxidized glucose. Each new measurement started withstandardization of the system using glucose (0.5-3.5 mM).

Preparation of Siloxanes for the Enzymatic Test

Preparation of the running buffer. The running buffer consists of (100mM) sodium phosphate, pH 7.0 in which (10 mM) 2.4 o-dianisidine wasdissolved. Preparation of oxygen carrier emulsion with glucose. In thecase when the PDMS copolymer was not water soluble a constantconcentration (0.5% w/w) of polypropylene glycol P2000 (PPG P2000) or upto 5% (w/w) Pluronic F 68 (trademark for a series nonionicsurface-active agents prepared by the addition of ethylene oxide to thepropylene glycols) as emulsifier was used. A given concentration of aPDMS copolymer with or without emulsifier was dissolved in the runningbuffer and sonicated for 2 minutes in Bronson water bath (40 mHz).Freshly prepared suspension was quickly distributed in 5 ml portionsinto labeled test tubes pre-filled with a defined glucose concentration.Before the injection into the enzyme thermistor (ET) system eachsuspension was saturated with pure oxygen for 2 minutes, and (100 μl)was injected into ET which was running at constant flow rate (0.7ml/min).

Reagents

A 25% aqueous solution of glutaraldehyde, Glucose oxidase (GO type X-S,from Aspergillus niger 208 U mg⁻¹) and 2.4 o-dianisidine were purchasedfrom Sigma Chemical CO (USA). Peroxidase (HRP) (250 U/mg) was obtainedfrom Biozyme Laboratories. Glucose, yeast extract and bactopeptone(digested casein) were obtained from Merck.(Darmstadt/Germany).Polypropylene glycol P2000 (PPG P2000) was a gift from MB-Sveda/Sweden,and Polydimethyl siloxane (PDMS) co-polymers were kindly obtained fromDow Corning (DC) USA supplied by the distributor in Belgium. Trosoperlcontrolled-pore glass (CPG) beads with free amino groups (particlediameter 125-140 nm, pore diameter 49.6 nm) were obtained from Schuller(Steinach, Germany). All solutions were prepared with phosphate buffer(sodium phosphate dibasic with sodium phosphate monobasic) 0.1 mol l⁻¹at pH 7.0.

Organisms

Bacillus thuringiensis, a laboratory strain and Streptomyces coelicolorA3(2) were used in these studies. The Bacillus thuringiensis wasmaintained at 4° C. on LB agar slants, and the inoculum was built bytransferring one loop of cells from the agar slant to 100 ml of liquidLB media (500 ml flask). The LB media consist of; yeast extract-0.5%,bactopeptone-1% and NaCL-1%, pH 7.0. Streptomyces coelicolor A3(2) wasmaintained at 4° C. on protein fraction extract (PFE) agar plate andspores were used for preparing a PFE based liquid inoculum.

Cultivation Conditions

The growth was carried out in the 3 L Erlenmeyer flasks filled with LB(1 litre) growth media and supplemented with 0.01-0.1% (w/w) of PDMScopolymer (DC Q2-5247) for Bacillus and 0.1-5% for Streptomyces and ascontrol no oxygen carrier was added. Each flask was closed with a tightstopper jointed to a gas filter. The gas filters were connected with aplastic tube to a sterile filter and further joined with the main airsupply via an Erlenmeyer flask filled with water sacking bottlesaturated under controlled flow of air. To keep the cultivation bottlesunder controlled temperature and agitation, during the growth, they wereplaced in a water bath with a set temperature and agitation speed.During defined hours by using a sterile syringe up to 50 ml of cellsuspension was drawn out for analysis.

Cell count The cell count was carried out for the bacillus cell culture.The samples were serially diluted with saline solution (0.9% NaCl). Theappropriately diluted samples (0.1 ml) were plated on LB agar plates andincubated at 30° C. for 24 h to form fully developed colonies.

Pigmented Actinorhodin determination Actinorhodin production has beenchecked out as follows: a known volume of liquid medium with growingbacteria was mixed with 2M KOH and left for 30 min., then centrifuged(10 min., 20000 g). The Actinorhodin content was measured in thesupernatant. Absorbance at λ=550 nm was followed by a Hitachi U-3200UV/VIS spectrophotometer (Kieser et al., 2000).

Screening of PDMS Copolymers by Using Glucose Oxidase/PeroxidaseThermosensor

A few commercially available Dow Corning (DC) PDMS co-polymers (table 1)were chosen and screened for their potential to increase the oxidationrate of glucose by using a co-immobilised glucose oxidase/peroxidasewhere the registration of the enzymatic reaction was combined with thethermal unit known as enzyme thermistor (ET). This study has alreadyindicated that such chemicals have a high potential for increasingoxygen solubility in water and do not kill the enzymes or microbialactivity. However, guidelines regarding the proper choice of acommercial product like the size and the proportion of the inbuiltblocks, its capacity to carry oxygen from water to the enzyme ormicrobial system are missing. As it is shown in FIG. 1, the glucoseconcentration continues to increase 0.5-1 mM, an increase of reactionrate is observed as first order kinetics (phase 1). As the substrateconcentration increases from 1.5-2.0 mM, the increase in the reactionrate begins to slow down, and with a large substrate concentration from2.5-3.5 mM, no further change in velocity is observed, phase 2 enteringthe zero-order kinetics. The reasons of the zero-order kinetics in thecase of oxidases at this range of glucose is due to the limitedconcentration of dissolved oxygen.

Different PDMS copolymers (table 1) at concentration range between 0.5to 25% (w/w) were tested with glucose in the range between 0.5-3.5 mM.The oxidation rate of glucose gets satisfactorily improved when 2.0 mMglucose is combined with 15% of each PDMS copolymer listed in table 1.

The individual PDMS co-polymer was suspended in water based solutions.In some experiments the PDMS copolymers were combined withpolypropyleneglycol P2000 (PPG P2000) or with Pluronic F 68. The choiceof these substances was based on the structural similarity to the nonPDMS block (table 1). Without glucose, PPG P2000 and Pluronic F 68 atconcentration 1 and 5% (w/w) respectively, passed though glucose oxidasesensor without producing a heat signal. The PPG P2000 used at aconcentration of 1% (w/w) with 1.5 mM glucose was not effective toimprove glucose oxidation, while Pluronic F 68 used at a concentrationof 5% (w/w) has less capacity to improve oxygen solubility on its own.To see the role of the block ratio of PDMS on the oxidation of 2.5 mMglucose 15% (w/w) PDMS copolymers were tested (table 1). Another aspectwas to see how the increased concentration of PDMS moiety either presentin the copolymer structure, or added separately into the water-basedsolution affects the glucose oxidation rate (FIG. 2). The product DC1248 that is almost a pure PDMS (table 1) was tested in combination withPPG P2000 (bar 8), DC Q2-5247 (bars 3,4) and DC 1598 (bars 6,7) in FIG.2. The almost pure PDMS marked DC 1248, added in the amount of 5% (w/w)together with 15% either DC Q2-5247 or DC 1598 shows a neglectableeffect.

The effect of the non PDMS moiety in tested copolymers is shown in FIG.3. It is interesting that Pluronic F 68 at 5% (w/w) used together withDC 1598 (bars 4, 5) improves the oxygen carrying capacity of the DC1598, while similar combination with DC Q2-5247 (bars 2, 3) is noteffective at all (FIG. 3). The explanation of these results is thecritical role of the PDMS ratio to the EO/PO block. In spite of the factthat PDMS is the key carrier for oxygen, the EO/PO block is the limitingfactor for the oxygen carrying capacity.

Thus, a more than 2-fold increase in oxygen carrying capacity wasobserved with the product containing only 18% DMS (Q2-5247) (FIG. 2, bar2). Addition of DC 1248 (DMS 96%) to the stimulatory Q2-5247, inincreasing proportion, had no effect (FIG. 2, bars 3 and 4). The sameresult was seen when DC 1248 was added to a copolymer with a higher(33%) DMS content (DC 1598) (FIG. 2, bars 5, 6, 7). The lowest oxygencarrying capacity was represented by DC 1248 (DMS content 96%) (FIG. 2,bar 8).

It was of interest to compare the most effective copolymer Q2-5247 withthe commercially important oxygen carrier perfluorodecalin. Thiscopolymer improved glucose oxidation by 75% compared to perfluorodecalin(FIG. 4, bars 4 and 2, respectively).

Thus, it has been demonstrated that PDMS copolymers can be an attractivealternative to perflurodecalin, for improving oxygen dependent enzymaticreactions in vitro and in vivo.

Effect of a PDMS Copolymer on Model Bacteria

PDMS copolymer marked Q2-5247 was tested for its effect onmultiplication of cells by Bacillus thuriginensis or antibioticproduction by Streptomyces coelicolor. In case of the Bacillusthuriginensis strain, the optimal final concentration of the PDMScopolymer used was 0.05% (w/w) (FIG. 5.). Higher concentrations were notsatisfactory, data not shown here. The model strain was chosen due tothe fact that it is a representative anaerobic strain and also that thisparticular species is a producer of a biopesticide. In this work we havenot measured the level of the biopesticide, but one could expect thatits level could be also increased. In some cases the improved oxygensolubility is not expressed in the form of an increased mass but in formthe of an increased level of metabolites (Ziomek et al., 1991, Elibol2001). Similar to Elibol (2001), we tested Q2-5247 for its potential toimprove the actinorhodin, an antibiotic product by Streptomycescoelicolor (A3). The results from the blue-pigmented antibioticactinorhodin are shown in table 2. In this model study the optimalconcentration of the used PDMS polymer, added at the beginning of theculture growth is 0.1% (w/w).

In the microbial system described in the literature, perfluorodecalin asoxygen carrier is more often used than PDMS copolymers, in spite of thefact that it is very expensive and it has to be used in highconcentrations in microbial systems to improve a biological process.Moreover, using perfluordecalin to improve fermentation yield, highwaste loads are created which might be problematic for the naturalbioremediation system. TABLE 1 The distribution of the main componentsin the polymerised blocks of the commercially available Dow Corning (DC)polydimethylsiloxanes (PDMS) which were tested with glucose oxidase isshown below. Dimethyl Product siloxane E0 PO name (%) (%) (%) MwViscosity DC 1248 96 0 2 3100 170 DCQ2-5247 18 35 46 27900 2305DCQ2-5573 19 35 46 58047 4450 DC 5604 24 50 26 6700 300 DC 1598 33 44 239590 548 DC 3581 95 4 1 31282 7500 DC 3580 17 1 83 5105 312E0 = ethylene oxidePO = propylene oxide

TABLE 2 The production of actinorhodin by Streptomyces coelicolor A3 (2)on the protein fraction extract (PFE*) based growth medium without andwith addition of PDMS copolymer marked DC Q2-5247 . The data areexpressed in OD units measured at λ 550 nm followed in the supernatantfrom the growth culture. Sampling without time PDMS 0.1% (w/w) PDMS 0.5%(w/w) PDMS (h) (OD) (OD) (OD) 60 0.11 0.20 0.13 84 0.12 0.33 0.17 1320.13 0.31 0.17 156 0.27 0.36 0.24*PFE protein fraction extract

1. A method for supplying oxygen to a water purification process, saidmethod comprising: a) providing an oxygen carrier of at least onecopolymer of dimethylsiloxane, ethylene oxide and propylene oxide; b)adding said oxygen carrier to the water purifying process; and c)contacting said oxygen carrier with an oxygen containing gas.
 2. Amethod according to claim 1, wherein said copolymer is added as anemulsion, or as a copolymer immobilized on and/or in a support.
 3. Amethod according to claim 2, wherein said support immobilized copolymerfurther includes immobilized microorganisms thereon.
 4. A methodaccording to claim 2 or 3, wherein said support is selected from thegroup consisting of organic supports and non-organic supports.
 5. Amethod according to claim 1, wherein said oxygen containing gas is addedto the process either continuously or batch-wise.
 6. A method accordingto claim 1, wherein said copolymer is added to the aerobic steps of thewater purifying process.
 7. A method according to claim 1, wherein saidat least one copolymer comprises 10-40% by weight of dimethylsiloxane,20-60% by weight of ethylene oxide, and 10-60% by weight of propyleneoxide.
 8. A method according to claim 7, wherein said copolymercomprises 15-35% by weight of dimethylsiloxane, 25-45% by weight ofethylene oxide and 20-50% by weight of propylene oxide.
 9. Use of atleast one copolymer of dimethylsiloxane, ethylene oxide and propyleneoxide, as an oxygen carrier in a water purification process.
 10. Useaccording to claim 9, wherein said at least one copolymer comprises10-40% by weight of dimethylsiloxane, 20-60% by weight of ethyleneoxide, and 10-60% by weight of propylene oxide.
 11. Use according toclaim 10, wherein said copolymer comprises 15-35% by weight ofdimethylsiloxane, 25-45% by weight of ethylene oxide and 20-50% byweight of propylene oxide.